Substituted liposaccharides useful in the treatment and prevention of endotoxemia

ABSTRACT

Novel substituted liposaccharides useful as in the prophylactic and affirmative treatment of endotoxemia including sepsis, septicemia and various forms of septic shock and methods of using these agents are provided. Also provided are methods of preparing these agents and intermediates useful therein.

RELATED APPLICATIONS

This application is a continuation-in-part of Ser. No. 08/461,675, filedJun. 5, 1995 now U.S. Pat. No. 5,750,664.

FIELD OF INVENTION

This invention relates to compounds which are useful as in theprophylactic and affirmative treatment of endotoxin exposure includingsepsis, septicemia, endotoxemia and various forms of septic shock.

BACKGROUND OF THE INVENTION

The invention relates to analogs of Lipid A which are useful asinhibitors of endotoxemia.

The incidence of gram negative bacteremia in the United States has beenestimated to be approximately 100,000 to 300,000 cases per year, with amortality rate of 30-60%. Antibiotics are commonly used as the primarychemotherapy for this disease; however, their bactericidal action canresult in disruption of the bacterium and concomitant release ofendotoxin, i.e., the lipopolysaccharide (LPS) moiety of the bacterialouter membrane. The liberated LPS induces a number of pathophysiologicalevents in mammals (collectively referred to as gram-negative endotoxemiaor sepsis syndrome). These include fever, generalized inflammation,disseminated intravascular coagulation (DIC), hypotension, acute renalfailure, acute respiratory distress syndrome (ARDS), hepatocellulardestruction and cardiac failure.

Although endotoxin initiates septic shock, it has little or no directtoxic effect on tissues; instead, it triggers an immunobiologicalresponse leading to a cascade of release of cytokines such astumor-necrosis factor (TNF), interleukin-1, interleukin-6 andinterleukin-8, and other biological mediators such as nitric oxide, aswell as an array of secondary mediators (e.g., prostaglandins,leukotrienes, interferons, platelet-activating factor, endorphins andcolony-stimulating factors). Generation of pathophysiologicalconcentrations of these cytokines and inflammatory mediators influencevasomotor tone, microvascular permeability and the aggregation ofleukocytes and platelets causing a syndrome termed systemic inflammatoryresponse syndrome (or SIRS) and septic shock.

The bacterial lipopolysaccharide molecule has three main regions: a longchain polysaccharide (O Antigen), a core region and a Lipid A region.The entire lipopolysaccharide molecule, as well as some of itsindividual components possess toxic effects described above. Most ofthese toxic effects, however, are believed to be attributable to theLipid A portion. Structurally, Lipid A is composed of a diphosphorylateddisaccharide acylated by long chain fatty acids.

Therapies for endotoxin-related diseases have generally been directedtowards controlling the inflammatory response. Such therapies includecorticosteriod treatment, suggested to ameliorate endotoxin-mediatedcell membrane injury and to reduce production of certain biologicalmediators; administration of antibodies designed to neutralize bacterialLPS; treatment with agents to suppress hypotension or with naloxonewhich apparently blocks the hypotensive effects associated with sepsissyndrome; and treatment with nonsteroidal anti-inflammatory drugs,purported to block cyclooxygenanses and thereby decrease the productionof certain secondary mediators such as prostaglandins and thromboxane.

However, none of these therapies to date has resulted in significantreduction in the morbidity and mortality resulting from sepsis andseptic shock syndrome. Thus there is a long felt need for agents toaffirmatively treat this disorder.

Christ, et al., "Anti-Endotoxin Compounds," U.S. Ser. No. 07/935,050,filed Aug. 25, 1992, the contents of which are included by reference,disclose certain disaccharide compounds, such as B531 shown below,useful for the treatment of endotoxemia. ##STR1##

Other references which disclose certain lipodisaccharides includeMacher, et al., Great Britain patent 2,179,945, Meyers, et al., GreatBritain patent 2,220,211, Shiba, et al., European patent 172,581,Anderson, et al., U.S. Pat. No. 4,495,346 and Shiba, et al., U.S. Pat.No. 5,066,794.

SUMMARY OF THE INVENTION

The present invention is directed to the treatment of sepsis, septicshock, endotoxemia and related disorders using novel liposaccharideanalogs. The compounds of the present invention possess advantages forpharmaceutical use such as enhanced pharmacological selectivity,efficacy, and in particular increased persistence of action. Arepresentative compound of this invention, compound 1, is shown below:##STR2##

Further, the present invention is directed to the prophylactic andaffirmative treatment of any LPS-mediated disorder. These disordersinclude, but are not limited to, sepsis, septicemia (including but notlimited to endotoxemia), endotoxemia resulting from gram negativebacteremia (with its accompanying symptoms of fever, generalizedinflammation, disseminated intravascular coagulation, hypotension, acuterenal failure, acute respiratory distress syndrome, adult respiratorydistress syndrome (ARDS), hepatocellular destruction and/or cardiacfailure) and various forms of septic shock (including but not limited toendotoxic shock). Also, compounds of this invention will be useful inthe prophylactic or affirmative treatment of localized or systemicinflammatory response to infection by different types of organisms,including gram negative bacteria, and in diseases related totranslocation of gram negative bacteria or endotoxin from the gut.

Together these disorders are termed systemic inflammatory responsesyndrome or SIRS (For a discussion of these terms, see Bone, et al.,Chest 1992; 101: 1644-55).

Definitions

In accordance with the present invention and as used herein, thefollowing terms, are defined with the following meanings, unlessexplicitly stated otherwise.

The term "alkyl" refers to aliphatic organic groups which may bebranched or straight and which may be optionally substituted with one ormore halogen atoms at any position along the alkyl chain. Alkyl groupsinclude both groups which are have a single unoccupied valence, forexample, --CH₂ --CH₃ and alkylene groups which have two unoccupiedvalences, for example --CH₂ --CH₂ --. As is obvious to those skilled inthe art, the single or double unoccupied valence will be used asappropriate to describe compounds which are chemically stable.

The term "prodrug" as used herein refers to any compound that has lessintrinsic activity than the "drug" but when administered to a biologicalsystem generates the "drug" substance either as a result of spontaneouschemical reaction or by enzyme catalyzed or metabolic reaction.Reference is made to various prodrugs such as acyl esters, carbonates,phosphates and urethanes, included herein as examples. The groupsillustrated are exemplary, not exhaustive and one skilled in the artcould prepare other known varieties of prodrugs. Such prodrugs of thecompounds of Formula I fall within the scope of the present invention.

The term "pharmaceutically acceptable salt" includes salts of compoundsof Formula I derived from the combination of a compound of thisinvention and an organic or inorganic acid or base. The compounds ofFormula I are useful in both non-ionized and salt form. In practice theuse of salt form amounts to use of base form; both forms are within thescope of the invention.

The term "geometric isomers" refers to "trans" or "cis" (or "entgegen"or "zusammen") isomers as generally understood by those skilled in theart. All geometric isomers are within the scope of the invention.

Further, compounds of the present invention may contain asymmetriccarbon atoms and hence can exist as stereoisomers, both enantiomers anddiastereomers. All stereoisomers and mixtures thereof are considered tofall within the scope of the present invention. The synthetic examplescited herein provide the most preferred isomer. It is evident that inaddition to the sugar moiety, additional asymmetric carbons may bepresent in compounds of Formula I, for example being present in the sidechains. In this event, all of the resulting diastereomers are consideredto fall within the scope of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the inhibition of release of TNF-α by Compound 1illustrating the inhibition of LPS-mediated induction of tumor necrosisfactor (TNF) in human whole blood by a compound of this invention.

FIG. 2 depicts the general scheme used to analyze antagonistic efficacyof drug after incubation in whole blood for various times.

FIG. 3 depicts the relationship between time versus ability of the testcompound to inhibit TNF-α and demonstrates that Compound 1 has asuperior duration of action as an LPS antagonist than does B531. Thesedata are the average of 7 separate experiments each run in triplicate.

DETAILED DESCRIPTION OF THE INVENTION Novel Liposaccharides

In one aspect, the present invention relates to the novel use ofsubstituted liposaccharides which comprise compounds of the generalformula I. ##STR3## where R¹ is selected from the group consisting of##STR4## where each J, K and Q, independently, is straight or branchedC1 to C15 alkyl; L is O, NH, or CH₂ ; M is O or NH; and G is NH, O, S,SO, or SO₂ ;

R² is straight or branched C5 to C18 acyl;

R³ is selected from the group consisting of

straight or branched C5 to C15 alkyl, ##STR5## where E is NH, O, S, SO,or SO₂ ; each A, B and D, independently, is straight or branched C1 toC15 alkyl;

R⁴ is selected from the group consisting of straight or branched C4 toC20 alkyl, and ##STR6## where each U and V, independently, is straightor branched C2 to C15 alkyl and W is hydrogen or straight or branched C1to C5 alkyl;

R_(A) is R⁵ or R⁵ --O--CH₂ --, R⁵ being selected from the groupconsisting of hydrogen, J',--J'--OH,--J'--O--K',--J'--O--K'--OH, and--J'--O--PO(OH)₂, where each J' and K', independently, is straight orbranched C1 to C5 alkyl;

R⁶ is selected from the group consisting of hydroxy, halogen, C1 to C5alkoxy and C1 to C5 acyloxy;

A¹ and A², independently, are selected from the group consisting of##STR7## where Z is straight or branched C1 to C10 alkyl; andpharmaceutically acceptable salts thereof.

Embodiments of the above formula include the following or combinationsof the following:

R² is C8 to C15 straight or branched alkyl;

R² is C9 to C12 straight or branched alkyl;

R² is C10 straight or branched alkyl;

A¹ and A², independently, are OH or --O--PO(OH)₂ ;

R⁶ is hydroxy;

R⁵ is C1 to C5 straight or branched alkyl;

R¹ is selected from the group consisting of ##STR8## where each J, K andQ, independently, is straight or branched C1 to C15 alkyl;

R³ is selected from the group consisting of ##STR9## where each A, B andD, independently, is straight or branched C1 to C15 alkyl;

the double bonds of R³ are cis or zusammen;

the double bonds of R³ are trans or entgegen;

R⁴ is selected from the group consisting of straight or branched C4 toC20 alkyl, and ##STR10## where U is straight or branched C2 to C5 alkyl,V is straight or branched C5 to C12 alkyl, and W is hydrogen or straightor branched C1 to C5 alkyl;

R_(A) is R⁵ ; and R_(A) is R⁵ --O--CH2--.

In other embodiments, each A¹ and A², independently, is selected fromthe group consisting of OH and --O--PO(OH)₂ ;

R¹ is selected from the group consisting of ##STR11## where each J, Kand Q, independently, is straight or branched C1 to C15 alkyl;

R² is straight or branched C8 to C15 alkyl;

R³ is selected from the group consisting of ##STR12## where each A, Band D, independently, is straight or branched C1 to C15 alkyl;

R⁴ is ##STR13## where U is straight or branched C2 to C5 alkyl, V isstraight or branched C5 to C12 alkyl and W is hydrogen or straight orbranched C1 to C5 alkyl;

and R⁵ is straight or branched C1 to C5 alkyl; and

R⁶ is hydroxy.

In another embodiment, A¹ and A² are --O--PO(OH)₂ ;

R¹ is selected from the group consisting of ##STR14## where each J andQ, independently, is straight or branched , C1 to C5 alkyl, and K isstraight or branched C8 to C15 alkyl;

R² is straight or branched C8 to C15 alkyl;

R³ is ##STR15## where A is straight or branched C5 to C12 alkyl and B isstraight or branched C6 to C12 alkyl;

R⁴ is ##STR16## where U is straight or branched C2 to C5 alkyl, V isstraight or branched C5 to C12 alkyl and W is hydrogen or straight orbranched C1 to C5 alkyl; and

R⁵ is straight or branched C1 to C5 alkyl; and

R⁶ is hydroxy.

In another embodiment, A¹ and A² are --O--PO(OH)₂ ;

R¹ is selected from the group consisting of ##STR17## where each J andQ, independently, straight or branched is C1 to C3 alkyl, and K isstraight or branched C10 to C12 alkyl;

R² is straight or branched C9 to C12 alkyl;

R³ is ##STR18## where A is straight or branched C8 to C12 alkyl and B isstraight or branched C6 to C10 alkyl;

R⁴ is ##STR19## where U is straight or branched C2 to C4 alkyl, V isstraight or branched C5 to C10 alkyl and W is hydrogen or straight orbranched C1 to C3 alkyl; and

R⁵ is straight or branched C1 to C3 alkyl; and

R⁶ is hydroxy.

In another embodiment, A¹ and A² are --O--PO(OH)₂ ;

R¹ is ##STR20## R² is (CH₂)₉ CH₃ ; R³ is ##STR21## R⁴ is ##STR22## R⁵ is--CH₃ ; and R⁶ is hydroxy.

Also within the scope of the invention are compounds in which R1 and R3are sulfonyls, i.e., compounds in which the carbonyl on these sidechains is replaced with SO₂. These compounds could be prepared bytreating the appropriately substituted alcoholic sugar with theappropriate alkylsulfonyl chloride. Thus R1 and R3 may also be selectedfrom the following with A, B, D, E, J, K, L, Q. and M as defined above:##STR23## Further, within the scope of the invention are compounds inwhich the point of unsaturation within the R3 side chain is not a doubleor triple carbon-carbon bond but is an optionally substituted aromaticgroup, i.e., compounds in which R3 may have the following structure:##STR24## where E is NH, O, S, SO, or SO₂ ; each A is straight orbranched C1 to C15 alkylene; D is straight or branched C1 to C15 alkyl;F is H, --OT, NT¹ T², --CO2T, or phenyl wherein each of T, T¹, and T² isindependently selected from hydrogen or C1 to C5 alkyl; B is straight orbranched C1 to C15 alkyl;

In general, preferred are compounds where:

R¹ is selected from the group consisting of: ##STR25## where each J, Kand Q independently, is straight or branched C1 to C15 alkyl;

R² is straight or branched C8 to C12 alkyl;

R³ is selected from the group consisting of: ##STR26## where each A, Band D, independently, is straight or branched C1 to C15 alkyl;

R⁴ is ##STR27## where U is straight or branched C2 to C5 alkyl, V isstraight or branched C4 to C10 alkyl and W is hydrogen or straight orbranched C1 to C5 alkyl;

R⁵ is selected from the group consisting of: hydrogen, --J', and --J'OHwhere J' is C1 to C5 straight or branched alkyl;

R⁶ is selected from the group consisting of hydroxy, halogen, and C1 toC5 acyloxy;

each A¹ and A², independently, are selected from the group consistingof: OH and ##STR28## and pharmaceutically acceptable salts thereof.

Most preferred are compounds of formula 1 where:

R¹ is selected from the group consisting of: ##STR29## where J isstraight or branched C1 to C5 alkyl and K is straight or branched C9 toC14 alkyl;

R² is straight or branched C8 to C12 alkyl;

R³ is ##STR30## where A is straight or branched C6 to C12 alkyl and B isstraight or branched C4 to C8 alkyl;

R⁴ is ##STR31## where U is straight or branched C2 to C4 alkyl, V isstraight or branched C5 to C9 alkyl and W is hydrogen or straight orbranched C1 to C3 alkyl;

R⁵ is C1 to C3 straight or branched alkyl;

R⁶ is hydroxy;

A¹ and A² are ##STR32## and pharmaceutically acceptable salts thereof.

General Synthetic Methods

This invention is also directed to processes for preparing compounds ofFormula I. Disclosed herein are general synthetic routes for preparingvariously substituted compounds of this invention. The synthesis for acompound of this invention, compound 1, is shown below.

Most of the reagents and starting materials are well known to thoseskilled in the art. Certain reagents and starting materials for thispreparation are described in detail by Christ, et al., in U. S.application Ser. No. 07/935,050 which will issue as U.S. Pat. No.5,530,113, the disclosure of which is hereby incorporated by reference.

One synthesis of the compounds of this invention is outlined below.Although this example describes the preparation of compound 1, use ofalternate starting materials will yield other analogs of this invention.Thus the synthesis is indeed general in nature.

For example, use of alternative alkylating agents in synthetic step 22will provide analogs with structurally differing substituents at R1.

The substitution pattern at R2 is controlled by the use of the properalkylating agent in step 15.

Further, substitution of suitable alternative compounds in step 25 inthe synthesis will produce analogs which differ with respect to R3.

Analogs without the oxygenated side chain at R_(A) may be prepared byusing slight variations in the synthetic scheme shown below as is willknown to those skilled in the art. For the compound in which R_(A) ismethyl, for example, the product of synthetic step 8, the tosylate,could have this leaving group replaced by iodine in the Finklesteinreaction. The iodo compound could be dehalogenated by treatment withzinc metal to give a methyl group at position R_(A).

A representative synthesis of the R4 side chain is outlined below. Thepreparation of variations of this side chain may be achieved byreplacing the starting material with other suitable starting materials.For example, the length or branching of this side chain may be preparedby starting with the appropriate starting material. Thus the use ofalternative tosylates in step 6 will produce variation in R4. ##STR33##

Thus the synthesis briefly outlined below provides versatile pathways tothe compounds of this invention. (For details regarding the synthesis,see the following experimental examples.) ##STR34##

Applicants believe that the above route, Route 1, is the superior methodof preparing compounds of the present invention due to a variety offactors such as use of cheaper starting materials, higher yields and theuse of less toxic chemical agents, the route illustrated below, Route 2,may be used to prepare compounds of this invention.

Most of the reagents and starting materials are well known to thoseskilled in the art. Certain reagents and starting materials for thispreparation are described in detail by Christ, et al., in U.S.application Ser. No. 07/935,050, the disclosure of which is herebyincorporated by reference. Although this example describes thepreparation of compound 1, use of alternate starting materials willyield other analogs of this invention. Thus the synthesis is indeedgeneral in nature.

For example, use of alternative alkylating agents in the preparation ofintermediate U will provide analogs with structurally differingsubstituents at R1.

The substitution pattern at R2 is controlled by the use of the properalkylating agent in the preparation of intermediate O.

Further, substitution of suitable alternative compounds for intermediateE in the preparation of intermediate G will produce analogs which differwith respect to R3.

A representative synthesis of the R4 side chain is outlined below. Thepreparation of variations of this side chain may be achieved byreplacing the starting material with other suitable starting materials.For example, the length or branching of this side chain may be preparedby starting with the appropriate starting material. (For detailsregarding the synthesis, see the following experimental examples.)##STR35##

A representative preparation of the "left" portion is outlined below.

A representative synthesis of the "right" portion of compound 1 is shownbelow. ##STR36##

These two "halves" of the molecule are then coupled as outlined belowand further elaborated to give compound 1. ##STR37##

Formulations

Lipid A analogs are administered in dosages which provide suitableinhibition of LPS activation of target cells; generally, these dosagesare, preferably between 0.01-50 mg/patient, more preferably, between0.05-25 mg/patient and most preferably, between 1-12 mg/patient. Mostpreferably the dosages are administered over three days as a continuousinfusion.

The term parenteral as used herein includes subcutaneous, intravenous,intramuscular, and intraarterial injections with a variety of infusiontechniques. intraarterial and intravenous injection as used hereinincludes administration through catheters. Preferred for certainindications are methods of administration which allow rapid access tothe tissue or organ being treated, such as intravenous injections forthe treatment of endotoxemia.

Pharmaceutical compositions containing the active ingredient may be inany form suitable for the intended method of administration.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadeaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension mayalso contain one or more preservative such as ethyl of n-propylp-hydroxybenzoate.

The pharmaceutical compositions of the invention are preferably in theform of a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents which have been mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,such as a solution in 1,3-butanediol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformation isotonic with the blood of the intended recipient; and aqueousand non-aqueous sterile suspensions which may include suspending agentsand thickening agents. The formulations may be presented in unit-dose ormulti-dose sealed containers, for example, ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders of the kindpreviously described.

It will be understood, however, that the specific dose level for anyparticular patient will depend on a variety of factors including theactivity of the specific compound employed; the age, body weight,general health, and sex of the individual being treated; the time androute of administration; the rate of excretion; other drugs which havepreviously been administered; and the severity of the particular diseaseundergoing therapy.

EXAMPLES

Examples of use of the method of the invention includes the following.The compounds of this invention and their preparation can be understoodfurther by the examples which illustrate some of the processes by whichthese compounds are prepared or used. These examples should not beconstrued as specifically limiting the invention and variations of theinvention, now known or later developed, are considered to fall withinthe scope of the present invention as hereinafter claimed.

Compounds of the present invention are referred to by compound number tothe tables below.

    __________________________________________________________________________                                        Formula 1    1 #STR38##    __________________________________________________________________________    Compound #          A.sup.1 /A.sup.2                     R.sup.1       R.sup.2    __________________________________________________________________________    1     OPO(OH).sub.2                     COCH.sub.2 CO(CH.sub.2).sub.10 CH.sub.3                                   (CH.sub.2).sub.9 CH.sub.3    2     OPO(OH).sub.2                     COCH.sub.2 CO(CH.sub.2).sub.10 CH.sub.3                                   (CH.sub.2).sub.9 CH.sub.3    3     OPO(OH).sub.2                     COCH.sub.2 CO(CH.sub.2).sub.10 CH.sub.3                                   (CH.sub.2).sub.9 CH.sub.3    4     OPO(OH).sub.2                     COCH.sub.2 CHOH(CH.sub.2).sub.10 CH.sub.3                                   (CH.sub.2).sub.9 CH.sub.3    5     OPO(OH).sub.2                     COCH.sub.2 CO(CH.sub.2).sub.10 CH.sub.3                                   (CH.sub.2).sub.9 CH.sub.3    6     OPO(OH).sub.2                     CO(CH.sub.2).sub.9 CH═CH(CH.sub.2).sub.5 CH.sub.3                                   (CH.sub.2).sub.9 CH.sub.3    7     OPO(OH).sub.2                     CO(CH.sub.2).sub.12 CH.sub.3                                   (CH.sub.2).sub.9 CH.sub.3    8     OPO(OH).sub.2                     COCH.sub.2 CH(O CH.sub.3)(CH.sub.2).sub.10 CH.sub.3                                   (CH.sub.2).sub.9 CH.sub.3    9     OPO(OH).sub.2                     COCH.sub.2 CH(O CH.sub.3)(CH.sub.2).sub.10 CH.sub.3                                   (CH.sub.2).sub.9 CH.sub.3    10    OPO(OH).sub.2                     COCH.sub.2 CH(OH)CH.sub.2).sub.10 CH.sub.3                                   (CH.sub.2).sub.9 CH.sub.3    11    OPO(OH).sub.2                     COCH.sub.2 CO(CH.sub.2).sub.10 CH.sub.3                                   (CH.sub.2).sub.9 CH.sub.3    __________________________________________________________________________    Compound #          R.sup.3       R.sup.4      R.sub.A                                          R.sup.6    __________________________________________________________________________    1     CO(CH.sub.2).sub.9 CH═CH(CH.sub.2).sub.5 CH.sub.3                        (CH.sub.2).sub.2 CH(O CH.sub.3)(CH.sub.2).sub.6                        CH.sub.3     CH.sub.2 OCH.sub.3                                          OH    2     CO(CH.sub.2).sub.9 CH═CH(CH.sub.2).sub.5 CH.sub.3                        (CH.sub.2).sub.2 CH(OH)(CH.sub.2).sub.6 CH.sub.3                                     CH.sub.2 OCH.sub.3                                          OH    3     CO(CH.sub.2).sub.16 CH.sub.3                        (CH.sub.2).sub.2 CH(OH)(CH.sub.2).sub.6 CH.sub.3                                     CH.sub.2 OCH.sub.3                                          OH    4     CO(CH.sub.2).sub.9 CH═CH(CH.sub.2).sub.5 CH.sub.3                        (CH.sub.2).sub.2 CH(OH)(CH.sub.2).sub.6 CH.sub.3                                     CH.sub.2 OCH.sub.3                                          OH    5     CO(CH.sub.2).sub.9 CH═CH(CH.sub.2).sub.5 CH.sub.3                        (CH.sub.2).sub.9 CH.sub.3                                     CH.sub.2 OCH.sub.3                                          OH    6     CO(CH.sub.2).sub.9 CH═CH(CH.sub.2).sub.5 CH.sub.3                        (CH.sub.2).sub.2 CH(OH)(CH.sub.2).sub.6 CH.sub.3                                     CH.sub.2 OCH.sub.3                                          OH    7     CO(CH.sub.2).sub.9 CH═CH(CH.sub.2).sub.5 CH.sub.3                        (CH.sub.2).sub.2 CH(OH)(CH.sub.2).sub.6 CH.sub.3                                     CH.sub.2 OCH.sub.3                                          OH    8     CO(CH.sub.2).sub.9 CH═CH(CH.sub.2).sub.5 CH.sub.3                        (CH.sub.2).sub.2 CH(O CH.sub.3)(CH.sub.2).sub.6                        CH.sub.3     CH.sub.2 OCH.sub.3                                          OH    9     CO(CH.sub.2).sub.9 CH═CH(CH.sub.2).sub.5 CH.sub.3                        (CH.sub.2).sub.2 CH(OH)(CH.sub.2).sub.6 CH.sub.3                                     CH.sub.2 OCH.sub.3                                          OH    10    CO(CH.sub.2).sub.9 CH═CH(CH.sub.2).sub.5 CH.sub.3                        (CH.sub.2).sub.2 CH(O CH.sub.3)(CH.sub.2).sub.6                        CH.sub.3     CH.sub.2 OCH.sub.3                                          OH    11    CO(CH.sub.2).sub.9 CH═CH(CH.sub.2).sub.5 CH.sub.3                        (CH.sub.2).sub.2 CH(O CH.sub.3)(CH.sub.2).sub.6                        CH.sub.3     CH.sub.3                                          OH    __________________________________________________________________________

CHEMICAL EXAMPLES

Unless otherwise noted, all reactions were conducted under an inertatmosphere. Intermediates and final products gave spectral analysis (forexample, nuclear magnetic resonance spectroscopy and/or massspectroscopy) consistent with their proposed structures. Reactions weremonitored by silica gel thin layer chromatography. Preparativechromatography, unless otherwise noted, was performed on silica gel.

Preparation of Compound 1 by Route 1

All sensitive reactions were run under nitrogen and in dry equipment andanhydrous sodium sulfate used as the drying agent unless otherwisespecified. All products gave satisfactory nuclear magnetic resonancespectra.

Purification of ##STR39##

The material (5 kg) was chromatographed on silica and eluted with agradient of hexane and EtOAc (100% to 33% hexane). The pure fractionswere combined and distilled (97-100° C. at 0.15 mm Hg). Yield ofpurified material 4,513 g. ##STR40##

To an ice cold solution of the ester (4500 g, 22.2 moles) in 12.6 L ofTHF was added sodium hydroxide (27 moles) in 10.8 L of water. Themixture was stirred briefly and 2.5 L of conc hydrochloric acid wasadded. The layers were separated and the aqueous layer re-extracted withEtOAc. The combined organic layers were washed with brine, dried oversodium sulfate and concentrated. The product slowly crystallized to give2983 g of white powder.

Purification of ##STR41##

To a solution of the acid (15.8 moles) in 33 L of acetonitrile was addeddicyclohexylamine (16.7 moles). The solution was heated to 60° C. andallowed to cool overnight. The crystals were collected and washed twicewith solvent and recrystallized from acetonitrile. To a suspension ofpreviously methanol washed Amberlite IR-120 Plus (12 kg) in EtOAc (24 L)and water (24 L) was added the above salt. The mixture was stirred forseveral hours and the organic layer separated. The aqueous layer wasre-extracted with EtOAc (12 L) and the combined organic layers weredried (sodium sulfate) and concentrated to give 2,997 g of a whitesolid. ##STR42##

To a hot (˜67° C.) 1M solution of lithium aluminum hydride (8 L) in THFwas slowly added a solution of the acid (1 kg) in 4 L of THF. Thesolution was allowed to cool overnight. The solution was slowly added to1M Aqueous HCl (5 L). The mixture was extracted with toluene (12 L). Theorganic layer was washed with sodium bicarbonate solution, dried (sodiumsulfate) and the solvent removed under vacuum to give a syrup which wasdistilled (103° C.) to give 914 g of a light yellow oil. ##STR43##

To a 0° C. solution of the diol (913.8 g) in pyridine (3 L) was added 3L of triethylamine, followed by a solution of tosyl chloride (1 kg) inpyridine (1.5 L) and triethylamine (1.5 L). The mixture was allowed towarm overnight and poured onto a cold solution of 6M aqueous HCl (16 L)and methylene chloride (8 L). The organic layer was separated and theaqueous layer extracted with additional methylene chloride. The combinedorganic layers were dried (sodium sulfate) and the solvent removed underreduced pressure. The residue was chromatographed twice on silica andeluted with a gradient of hexane:EtOAc (9:1 to 1:6) to give 642 g oftosylate. ##STR44##

To a suspension of 60% sodium hydride oil dispersion (8.68 moles) in1.15 L of DMF and 1.1 L of THF was slowly added the tosylate (1.139 kg)and methyl iodide (7.7 kg) in 1.15 L of DMF and 1.1 L of THF. Themixture was stirred overnight and then diluted with DMF (3 L) and slowlyadded to an satd aqueous solution of ammonium chloride. The mixture wasextracted with hexane (8 L) which was dried (sodium sulfate) and thesolvent removed to give a orange/brown oil. The oil was chromatographedon silica and eluted with a gradient (hexane:EtOAc 100:0 to 6:1) to give940 g of a light yellow oil. ##STR45##

To a suspension of the aminosugar (1019 g) in 5 L of MeOH was added a25% solution of NaOMe in MeOH (1080 mL, 5 moles), followed by 610 mL ofethyl trifluoroacetate. The mixture was stirred overnight and thesolvent removed under reduced pressure and the residue titurated withisopropanol. The mixture was filtered and the residue washed withadditional isopropanol to give 1369 g of product. ##STR46##

To a suspension of the hydroxy sugar (1300 g) in pyridine (4 L) wasadded dimethylaminopyridine (79 g), followed by acetic anhydride (2713mL). The mixture was stirred overnight. The solvent was removed underreduced pressure. Toluene (5×500 mL) was added and also removed underreduced pressure to give a solid which was chromatographed on silica.Elution with hexane:EtOAc (1:1) gave 1479 g of a white solid. ##STR47##

To a solution of the acetylated sugar (1479 g) in 8 L of methylenechloride was added allyl alcohol (764 mL), followed by slow addition oftin tetrachloride (976 mL). The mixture was stirred overnight and slowlypoured onto ice cold water (7.5 L). The organic layer was separated andthe aqueous layer washed with additional methylene chloride. Thecombined organic layers were washed with aqueous sodium bicarbonate,dried and concentrated under reduced pressure. The residue waschromatographed on silica (7.5 kg) and eluted with a hexane:EtOAcgradient (4:1 to 1:1) to give 1327 g of a pale yellow oil. ##STR48##

To a ice cold solution of protected sugar (1322 g) in 8.5 L of methanolwas added to a 25% solution of NaOMe in methanol (437 mL) over one hour.To this was added previously washed 1740 g of Amberlite IR-120 Plusresin. The mixture was filtered, concentrated and the residuechromatographed on silica. Elution with methanol gave 907 g of product.##STR49##

The triol was suspended in acetone (7.5 L) and camphorsulfonic acid (85g) was added and then 2,2-dimethoxypropane (965 mL) was slowly added.The mixture was stirred overnight followed by the addition oftriethylamine (51 mL). The solvent was removed under reduced pressure togive a brown solid which was chromatographed on silica. Elution with ahexane:EtOAc gradient (3:1 to 2:1) gave 842 g of a semi-white gum.##STR50##

To a suspension of 60% sodium hydride oil dispersion (82 g) in 2.2 L ofTHF and 580 mL of DMF and was added the tosylate (351 g) and a solutionof the free alcohol (400 g) in a mixture of 1360 mL of THF and 360 mL ofDMF. The mixture was stirred overnight. The mixture was cooled in iceand methanol was added, followed by water (2 L). The mixture wasextracted three times with EtOAc. The combined organic layers were driedand concentrated. The resulting mixture was chromatographed on silica.Gradient elution with hexane:EtOAc (19:1 to 1:1) gave 711 g. ##STR51##

To a mixture of 48% aqueous hydrofluoric acid in 1500 mL of acetonitrilein a Teflon bottle was added a solution of the starting material (613 g)in 750 mL of acetonitrile and 750 mL of methylene chloride. The mixturewas stirred for one hour and poured onto 8 L of water. The mixture wasextracted with methylene chloride (4×2 L). The combined organic layerswere washed with aqueous satd sodium bicarbonate solution, dried andconcentrated under reduced pressure. The residue was chromatographed onsilica. Gradient elution with methylene chloride:methanol (39:1 to 9:1)gave 519 g of product. ##STR52##

To a solution of the diol (577 g) in pyridine (5 L) was added tosylchloride (339 g) and N,N-dimethylaminopyridine (14.5 g). The mixture wasstirred at RT for two days and then poured onto 14 L of cold aqueous 1Mhydrochloric acid. The mixture was extracted (2×5 L) with methylenechloride. The combined organic layers were dried and concentrated. Theresidue was chromatographed on silica. gradient elution (hexane:EtOAc,6:1 to 1:1) gave 632 g of a yellow syrup which slowly crystallized onstanding. ##STR53##

To an 85° C. solution of 25% sodium methoxide in methanol (1825 mL) inDMF (1365 mL) was added the tosylate (714 g) in DMF (1365 mL) over 1.25hour. The mixture was stirred 30 minutes and cooled to 4° C. and pouredonto an ice cold mixture of aqueous 1M hydrochloric acid and 4.6 kg ofice. The mixture was stirred for 30 minutes and filtered. The filtratewas washed with 2 L of water and the combined aqueous layers wereextracted with 2×4 L of EtOAc. The combined organic layers were driedand concentrated. The residue was purified by chromatography on silica.Gradient elution (hexane:ethyl acetate 3:1 to 1:1) gave 549 g of a paleyellow to white solid. ##STR54##

This reaction was run under argon. To a solution of potassium t-butoxide(139 g) in 440 mL of DMSO was added a solution of the sugar (247 g) in440 mL of anhydrous DMSO. The mixture was heated to 85° C. for 1.5 hourand then 250 mL of water was added and the mixture heated overnight at85° C. and cooled in an ice bath. The mixture was poured onto 3.5 L ofbrine and the mixture extracted with 3×750 mL of methylene chloride. Thecombined organic layers were dried and concentrated to yield 560 g of abrown oil. ##STR55##

To a mixture of the free amine (199 g) 780 mL of THF and 390 mL of satdaqueous sodium bicarbonate was added Troc-Cl (157 g). After 1/2 hour,the mixture was slowly poured onto a solution of 500 mL of 40% aqueousmethylamine and 3 L of water. The mixture was extracted with 2×1750 mLof methylene chloride. The combined organic layers were dried andconcentrated. The residue was chromatographed on silica. Gradientelution with hexane: EtOAc (5:1 to 1:1) gave a quantitative yield of 287g of a yellow to off-white solid. ##STR56##

To a solution of the hydroxy-sugar in 2 L of methylene chloride wasadded tetrazole (155.6 g), followed by diallyidiisopropylphosphoramidite(182 mL). After 1/2 hour, the mixture was poured onto an ice coldmixture of Oxone® (potassium peroxymonosulfate) (455.6 g), water (1.25L) and THF (2.5 L). After 15 minutes, this mixture was poured onto cold10% aqueous sodium thiosulfate. After 15 minutes, the mixture wasextracted with 2 L of methylene chloride. The organic layer wasseparated, the aqueous layer re-extracted with methylene chloride andthe combined organic layers dried and the solvent removed under vacuum.The residue was chromatographed on silica. Gradient elution withhexane/ethyl acetate (6:1 to 2:1) gave 205.7 g of pale yellow syrup.##STR57##

To a solution of 48% aqueous hydrofluoric acid, 400 mL, in acetonitrile,1.2 L in a Teflon container was added a solution of the sugar, 138.8 g,in methylene chloride, 500 mL. The mixture was stirred overnight,diluted with water, 3 L, and extracted with methylene chloride, 2.4 L.The organic layer was washed with aqueous sodium bicarbonate solution,dried and the solvent was removed under reduced pressure. The residuewas chromatographed on silica. Gradient elution (hexane:ethyl acetate2:1 to 1:1), followed by elution with a gradient of methylene chloride:methanol (19:1 to 9:1) gave 129.2 g as a waxy gum. ##STR58##

To a ice cold solution of 450 g of 1-decanol in 685 mL of triethylamineand 1125 mL of methylene chloride was added 330 mL of mesyl chloride.The cooling bath was removed after 11/2 hour and the solvent removedunder reduced pressure. To the residue was added 2.5 L of 1M aqueoushydrochloric acid. This mixture was extracted 3×2 L of methylenechloride. The organic layers were combined, dried and the solventremoved under reduced pressure. The residue was chromatographed onsilica. Elution with 1:1 hexane:ethyl acetate gave 651 g of product.##STR59##

To a suspension of 60% sodium hydride mineral oil dispersion in 1 L ofTHF and 470 mL of DMF was added a solution of the alcohol in 280 mL ofDMF and 1 L of THF over 1 hour. The mesylate, 470 g, was then added over15 minutes. After 2 days, 400 mL of methanol was added, followed by 4 kgof ice and 4 L of water. This mixture was extracted with 2×4 L of ethylacetate. The combined organic layers were dried and the solvent wasremoved under reduced pressure. The residue was chromatographed onsilica. Gradient elution with hexane:EtOAc (39:1 to 2:1) gave 618 g.##STR60##

A solution of the sugar, 520 g, in 5.2 L of glacial acetic acid and 1.3L of water was stirred overnight. It was poured onto 7.5 L of water andfiltered. The filtrate was dried by azeotropic distillation with toluene(3×500 mL) under reduced pressure to give 458 g. ##STR61##

This reaction was run under argon. To a suspension of potassiumt-butoxide, 295 g, in DMSO, 1 L, was added a solution of 340 g of thesugar in 1.5 L of DMSO. The mixture was heated to 85° C. for 11/4 hourand 1.4 L of 3M aqueous potassium hydroxide was added and the mixturestirred overnight at 85° C. The mixture was cooled to room temperatureand poured onto a mixture of 3.5 L of brine and 3.5 L of water. Themixture was extracted three times with methylene chloride, the mixturedried and the solvent was removed under reduced pressure. The residuewas chromatographed on silica. gradient elution with methylenechloride:methanol 19:1 to 4:1) gave 740 g of product. ##STR62##

A solution of the aminosugar, 740 g, in benzophenone imine, 338 g, washeated at 45° C. overnight. The mixture was chromatographed on silicaand eluted with a gradient of hexane/ethyl acetate (39:1 to 1/1) to give371 g of a pale yellow solid. ##STR63##

To a solution of the diol sugar, 366 g, in 1.3 L of DMF was addedimidazole, 118 g, followed by t-butyldimethylsilyl chloride, 117 g.After 5 minutes, the mixture was poured onto 1.4 L of aqueous saturatedsodium bicarbonate. The mixture was extracted with ethyl acetate threetimes. The organic layers were combined, the solvent was removed underreduced pressure and the residue chromatographed on silica. Gradientelution with hexane/ethyl acetate (49:1 to 4:1) gave 446 g of a syrup.##STR64##

To a solution of the alcohol, 437 g, in toluene, 3 L, was addedpyridine, 225 mL and the solution cooled in an ice bath. Phosgene, 531mL of a 1.9M solution in toluene was added and the solution stirred for10 minutes. Allyl alcohol, 469 mL, was added. After 40 minutes,saturated aqueous sodium bicarbonate solution, 2.3 L, was added and themixture extracted with ethyl acetate. The organic layer was separated,dried and the solvent was removed under reduced pressure. The residuewas chromatographed on silica. Gradient elution with hexane/ethylacetate (49:1 to 4:1) gave 441 g of yellow syrup. ##STR65##

To a solution of the sugar, 431 g, in THF, 200 mL, was added glacialacetic acid, 330 mL, and water, 110 mL. The mixture was stirred forthree hours, cooled in ice and 6.6 L of 1M aqueous sodium hydroxide wasadded. The mixture was extracted with methylene chloride, 2×2 L. Thecombined organic layers were dried and the solvent was removed underreduced pressure. The residue was chromatographed on silica. Gradientelution with methylene chloride:methanol (19:1 to 4:1) gave the amine,309 g as a syrup. ##STR66##

To a ice cold solution of the amino sugar, 309 g, in 3 L of methylenechloride was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDC), 435 g, followed in 10 minutes by the carboxylicacid, 275 g. After 10 minutes, the mixture was extracted with saturatedaqueous sodium bicarbonate. The organic layer was separated, the aqueouslayer re-extracted with methylene chloride, the combined organic layersdried and the solvent was removed under reduced pressure. The residuewas chromatographed on silica. Gradient elution (hexane:ethyl acetate19:1 to 3:1) gave 338 g of pale yellow syrup. ##STR67##

To a solution of 48% aqueous hydrofluoric acid, 11 mL, in acetonitrile293 mL, was added 4.6 g of silica gel, followed by a solution of thesugar, 146.7 g, in methylene chloride, 147 mL. After one half hour, themixture was diluted with water, 975 mL, and extracted with methylenechloride. The organic layer was separated and the aqueous layerre-extracted with methylene chloride. The combined organic layers werewashed with aqueous sodium bicarbonate solution, dried and the solventwas removed under reduced pressure. The residue was chromatographed onsilica. Gradient elution (hexane:ethyl acetate 5:1 to 0:1) gave 110.4 gof an off-white waxy solid. ##STR68##

To a solution of the sugar, 129 g, in 500 g of trichloroacetonitrile wasadded potassium carbonate, 240 g. The mixture was stirred for one halfhour and filtered through diatomaceous earth. The filter cake was washedwith methylene chloride and the filtrates combined and the solvent wasremoved under reduced pressure. The residue was chromatographed onsilica. Gradient elution (hexane:ethyl acetate 1:1 to 0:1) gave 145.7 gof a yellow gum. ##STR69##

The left sugar, 145.7 g, and of the right sugar, 109.2 g, wereazetropically dried by evaporating toluene (3×200 mL). A solution of thetwo sugars in 750 mL of methylene chloride was added to an ice coldsolution of silver triflate, 62.7 g in 130 mL of methylene chloride. Themixture was warmed to room temperature and stirred overnight. Themixture was poured onto a mixture of saturated aqueous sodiumbicarbonate and sodium thiosulfate solution. The organic layer wasseparated and the aqueous layer washed with methylene chloride. Thecombined organic layers were dried and the solvent was removed underreduced pressure. The residue was chromatographed twice on silica.Gradient elution with hexane:ethyl acetate (5:1 to 1:1) gave 189.56 g ofa sticky foam. ##STR70##

To a solution of the disaccharide, 188.7 g, in THF, 590 mL, was addedzinc dust, 457.6 g, followed by glacial acetic acid, 395 mL. After onehalf hour, the mixture was filtered through diatomaceous earth and thefilter cake washed with THF. The organic layers were combined and thesolvent was removed under reduced pressure. The residue was driedazeotropically by distilled benzene from the residue (4×250 mL) to give223.1 g of a pink gum. ##STR71##

To a solution of the sugar, 223.1 g, in 1.3 L of THF was added asolution of sodium bicarbonate, 37.5 g, in 250 mL of water.cis-11-Octadecenoyl chloride, 67.4 g, was added. After 10 minutes, themixture was extracted twice with ethyl acetate. The combined organiclayers were dried and the solvent was removed under reduced pressure.The residue was chromatographed on silica. Gradient elution withhexane:ethyl acetate (2:1 to 0:1) gave 160.2 g of pale yellow wax.##STR72##

A solution of the sugar, 161.3 g, in methylene chloride, 215 mL, in aTeflon bottle was added to a solution of 48% hydrofluoric acid, 150 mL,in acetonitrile, 474 mL. After four hours, the mixture was poured onto500 mL of water. The mixture was extracted twice with methylenechloride. The combined organic layers were washed with aqueous saturatedsodium bicarbonate, dried and the solvent was removed under reducedpressure. The residue was chromatographed on silica. Gradient elution(methylene chloride:ethyl acetate:methanol 500:500:20 to 500:500:160)gave a yellow waxy gum. ##STR73##

The sugar, 719 mg, was dissolved in methylene chloride and sodiumsulfate (1.4 g) was added. Diallyidiiospropylphosphoramidite (189 μL)and tetrazole (162 mg) were added, the mixture stirred for 10 minutesand then cooled to -78° C. A solution of m-chloroperoxybenzoic acid (192mg) in methylene chloride (4 mL) was added dropwise. The mixture waswashed with aqueous sodium thiosulfate and with aqueous sodiumbicarbonate, dried (sodium sulfate) and the solvent removed underreduced pressure. The residue was chromatographed to give 660 mg.##STR74##

To a solution of tetrakis(triphenylphosphine)palladium (0) (166 mg) in 2mL of tetrahydrofuran: acetic acid (10:1) mixture was added a solutionof intermediate Z (660 mg) in 3 mL of the same solvent mixture. After 30minutes, additional tetrakis(triphenylphosphine)palladium (0) was added.After an additional 11/2 hours, toluene was added and the solventremoved under reduced pressure. The mixture was purified bychromatography on diethylaminoethylcellulose. The purified mixture wasdissolved in 0.1N aqueous sodium hydroxide, filtered through a 0.45 μsterile filter and purified by HPLC on a YMC-Pack ODS-AP column to give130 mg of compound 1.

Analytical data for compound 1 made by the methods described above isgiven below:

Compound 1: ¹ H NMR (CD₃ OD) δ: 5.3 (1H, m), 4.6 (1, m), 4.0 (m, m), 3.9(1H, d), 3.7 (1H, t), 3.6 (1H, t), 3.4 (3H, s), 3.3 (3H, t), 2.6 (2H,t), 2.3 (2H, m), 2.0 (2H, m), 1.7-1.2 (m, m), 0.9 (6H, t).

³¹ P NMR (CD₃ OD) δ: 4.71, 3.98.

Preparation of Compound 1 by Route 2 Preparation of Compound 1 Example 1

Intermediate B

To a suspension of intermediate A (15 g), prepared by the method ofChrist, et al., European patent application 92309057.5, in CH₂ Cl₂ (150mL) and 48% HBF₄ (29.2 g), cooled via ice-bath, was added TMSCHN₂ (165mL as a 2M solution in hexane). The mixture was stirred until thereaction was almost complete by TLC and then methanol (20 mL) was addedfollowed by acetic acid (10 mL). Aqueous sodium bicarbonate was addedand the mixture extracted with methylene chloride. The mixture was dried(sodium sulfate) and the solvent removed under reduced pressure.Chromatography of the residue gave B, 14.9 g.

Example 2

Intermediate C

To a cold (0° C.) solution of B (14.9 g) in methylene chloride (100 mL)was slowly added diisobutylaluminum hydride (140 mL as a 1M solution inhexanes) until reaction was complete as determined by TLC. The reactionwas quenched by the addition of aqueous 1N hydrochloric acid (100 mL)followed by conc. hydrochloric acid (50 mL). The layers were allowed toseparate and the aqueous layer was re-extracted with CH₂ Cl₂. Thecombined organic layers were then washed with brine, dried over sodiumsulfate and concentrated under reduced pressure. After purification bysilica chromatography, 12.06 g of intermediate C was obtained.

Example 3

Intermediate D

To a solution of C (10.64 g) in methylene chloride (40 mL) was addedtriethylamine (15.75 mL), p-toluenesulfonyl chloride (11.86 g) anddimethylaminopyridine (690 mg). The resulting suspension was allowed tostir until reaction was complete as determined by TLC then quenched viawater work-up with methylene chloride extraction. After purification bysilica chromatography, 18.7 g of D was obtained.

Example 4

Intermediate E

To a solution of D (18.7 g) in 200 mL of acetone was added sodium iodide(24.6 g). The mixture heated at reflux for 11/2 hours, the solventremoved under reduced pressure and the residue partitioned between waterand hexane. The organic layer was separated, dried (sodium sulfate) andthe solvent removed. Chromatography (silica) gave 15.4 g of E as acolorless liquid.

Example 5

Intermediate F

This compound was prepared by the method of Christ, et al., EuropeanPatent Application 92309057.5.

Example 6

Intermediate G

To a solution of 18.6 g of intermediate F and 15.4 g of intermediate Ein hexane was added 23.9 g of silver oxide and the mixture refluxedovernight. The mixture was cooled, filtered through diatomaceous earth,the solvent removed and the residue chromatographed (silica) to giveintermediate G (21 g) as a colorless syrup.

Example 7

Intermediate H

To a cold (0° C.) solution of intermediate G (21 g) in methylenechloride was added dropwise 3.5 mL of 48% tetrafluoroboric acid. After 5minutes, the mixture was washed with aqueous sodium bicarbonate solutionand with brine. The mixture was concentrated under reduced pressure andchromatographed (silica) to give intermediate H, 18.7 g, as a colorlesssyrup.

Example 8

Intermediate I

To a solution of intermediate H (17.6 g) in neat methyl iodide (105 mL)was added silver oxide (83 g). The mixture was stirred overnight andthen diluted with hexane and filtered through diatomaceous earth. Themixture was concentrated under reduced pressure and the residuedissolved in methylene chloride (40 mL). The mixture was cooled to 0° C.and to it was added imidazole (2.44 g) and t-butyldimethylsilyl chloride(4.7 mL). It was stirred overnight and 150 mL of sodium bicarbonatesolution was added. The organic layer was dried (sodium sulfate) andchromatographed (silica) to give intermediate I, 10.5 g, as a colorlesssyrup.

Example 9

Intermediate J

Intermediate I was dissolved in 100 mL of methylene chloride to which asadded diallyidiisopropylphosphoramidite (7.4 mL), followed by tetrazole(6.37 g). The mixture was cooled and stirred for 20 minutes. Asuspension of metachloroperoxybenzoic acid (24.2 mmol) in 50 mL ofmethylene chloride was added over 15 minutes while the temperature ofthe reaction was maintained below -60° C. Sodium bicarbonate solutionwas added and the organic layer separated, dried (sodium sulfate) andthe solvent removed under reduced pressure. Chromatography (silica) gave14 g of a colorless syrup of intermediate J.

Example 10

Intermediate K

To a suspension of 39.5 g of di(thiophenyl)tin (prepared by the methodof Christ, et al., European patent application 92309057.5) in 235 mL ofmethylene chloride was added thiophenol (12 mL). To this mixture wasadded triethylamine dropwise over 15 minutes. A portion (150 mL) of this"tin reagent" mixture was added dropwise over 15 minutes to a solutionof intermediate J (12.9 g) in 25 mL of methylene chloride. The remainderof the "tin reagent" was added over 30 minutes to drive the reaction tocompletion. The mixture was diluted with ethyl acetate and washed withaqueous 1N sodium hydroxide and with brine. The organic layer was dried(sodium sulfate), the solvent removed and the residue chromatographed togive 11.1 g of a yellow syrup, intermediate K.

Example 11

Intermediate L

To a cold solution of intermediate K (11.1 g) and pyridine (7.1 mL) in80 mL of methylene chloride was added trichloroethyl chloroformate (2.9mL) and the mixture was stirred overnight. Aqueous sodium bicarbonatesolution was added, the organic layer was separated, dried (sodiumsulfate) and the solvent removed under reduced pressure. Chromatographygave intermediate L, 12.96 g as light yellow solid.

Example 12

Intermediate M

Intermediate L, 12.96 g, was dissolved in methylene chloride. To thismixture was added a 6M solution of hydrogen fluoride in acetonitrile andthe mixture stirred for 4 hours. Aqueous sodium bicarbonate solution wasadded the organic layer separated, dried (sodium sulfate) and thesolvent removed under reduced pressure. Chromatography gave 10.9 g of anamber syrup, intermediate M.

Example 13

Intermediate N

To a solution of intermediate M (9.5 g) in 50 mL oftrichlororacetonitrile was added potassium carbonate (15 g) and themixture stirred for 10 minutes. The mixture was filtered throughdiatomaceous earth and the solvent removed under reduced pressure.Chromatography gave 14.5 g, intermediate N which was used at once inExample 19.

Example 14

Intermediate O

To a solution of intermediate F (160 g) in hexane (475 mL) andiododecane (474 mL) was added silver oxide (723 g). The mixture washeated at 70° C. in the dark for 2 hours and filtered throughdiatomaceous earth. The solution was concentrated under reduced pressureand the residue chromatographed to give 221 g of intermediate O as acolorless oil.

Example 15

Intermediate P

To a solution of intermediate O (30 g) in methylene chloride (90 mL) andacetonitrile (90 mL)was added a solution of 48% aqueous hydrogenfluoride (9 mL) in acetonitrile (81 mL). The mixture was stirred for 30minutes and 350 mL of aqueous sodium bicarbonate was added. The mixturewas extracted with methylene chloride. The organic layer was dried(sodium sulfate), the solvent removed under reduced pressure and theresidue chromatographed to yield 30 g of intermediate P as a yellow oil.

Example 16

Intermediate Q

To a cold (0° C.) solution of intermediate P (30 g) and imidazole (10.2g) in methylene chloride (500 mL) was added t-butyldimethylsilylchloride (10.85 g). The mixture was stirred for 11/2 hours and thenpoured onto 400 mL of saturated aqueous ammonium chloride. The organiclayer was separated, dried (sodium sulfate), the solvent removed underreduced pressure and the residue chromatographed to give 34.5 g ofintermediate Q as a colorless syrup.

Example 17

Intermediate R

To a cold (0° C.) solution of intermediate Q (32.2 g) and pyridine (184mL) in toluene (213 mL) was added a 1.94M solution of phosgene intoluene. After 20 minutes, allyl alcohol (31 mL) was added and themixture stirred for 30 minutes. Aqueous sodium bicarbonate was added,the organic layer separated, dried (sodium sulfate) and the solventremoved under reduced pressure. Chromatography gave 36.9 g ofintermediate R as a colorless syrup.

Example 18

Intermediate S

To a solution of 2.4 mL of 48% aqueous hydrogen fluoride in 48 mL ofacetonitrile was added a solution of intermediate R (20 g) in methylenechloride (24 mL) and the mixture stirred overnight. Aqueous sodiumbicarbonate solution was added, the organic layer separated and dried(sodium sulfate) and the solvent removed under reduced pressure.Chromatography yielded 11 g of intermediate S as a colorless syrup.

Example 19

Intermediate T

Intermediate S (8.97 g) and intermediate N (14.5 g) were dissolved intoluene (20 mL) and the mixture dried by azeotropic removal of thesolvent. This procedure was repeated three times. The dried mixture wasdissolved in 50 mL of methylene chloride which was slowly added to asolution of silver triflate (5.8 g) in 50 mL of methylene chloride. Themixture was stirred for 10 minutes and 250 mL of aqueous sodiumbicarbonate solution and 250 mL of 10% aqueous sodium thiosulfate wasadded. The organic layer was separated, dried (sodium sulfate) and thesolvent removed under reduced pressure. Chromatography gave 13 g ofintermediate T as a pale yellow syrup.

Example 20

Intermediate U

To a solution of intermediate T in methylene chloride (10 mL) was slowlyadded tin(II)tris-benzenethiolate triethylamine complex (12 mL of a 0.5Msolution in methylene chloride). After 10 minutes, an additionalequivalent of tin reagent was added. After an additional 15 minutes, anadditional equivalent was added. After 15 minutes, ethyl acetate (250mL) was added and the mixture extracted with 1N aqueous sodium hydroxidesolution (250 mL). The mixture was dried (sodium sulfate) andconcentrated under reduced pressure. Toluene was added and the solventremoved under reduced pressure to give an oil which was used in the nexttransformation without further purification.

Example 21

Intermediate V

To a cooled (0° C.) solution of intermediate U (2 mmol) in methylenechloride (5 mL) was added 3-ketotetradecanoic acid (997 mg) , preparedby the method of Christ, et al., European patent application 92309057.5,followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(1.5 g) and the mixture stirred for approximately 30 minutes. Themixture was diluted with methylene chloride (150 mL), washed with 1Naqueous sodium hydroxide, dried (sodium sulfate) and the solvent removedunder reduced pressure. Chromatography on silica followed bychromatography on basic alumina gave 1.64 g of intermediate V.

Example 22

Intermediate W

A solution of intermediate V (1.45 g) in glacial acetic acid (5 mL) wasadded to a suspension of well stirred zinc copper couple (14 g) inacetic acid (10 mL). The mixture was stirred for 15 minutes andadditional zinc/copper couple (10 g) was added. After an additional 15minutes, the mixture was filtered through diatomaceous earth which wasthen washed with ethyl acetate. The combined washings were diluted withtoluene and the solvent removed under reduced pressure. The residue waschromatographed on a bilayered mixture of basic alumina and silica togive intermediate W which was used without further purification.

Example 23

Intermediate X

A solution of intermediate W (1.02 mmol) and cis-vaccenic acid (575 mg)was dissolved in toluene (5 mL) three times and the solvent removedunder reduced pressure. The dried residue was dissolved in methylenechloride (3 mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (780 mg) was added and the mixture stirred for 3 hours.The mixture was diluted with methylene chloride and chromatographeddirectly to give 734 mg of intermediate X. Further chromatography of theimpure fractions gave an additional 58 mg of material.

Example 24

Intermediate Y

To a solution of intermediate X (785 mg) in methylene chloride (10 mL)was added a solution of 48% aqueous hydrogen fluoride in acetonitrile(15 mL). The mixture was stirred for 90 minutes, diluted with methylenechloride (50 mL), washed with water, and with aqueous sodium bicarbonatesolution. The mixture was dried (sodium sulfate) and chromatographed togive intermediate Y, 719 mg.

Example 25

Intermediate Z

Intermediate Y (719 mg) was dissolved in methylene chloride and sodiumsulfate (1.4 g) was added. Diallyidiiospropylphosphoramidite (189 μL)and tetrazole (162 mg) were added, the mixture stirred for 10 minutesand then cooled to -78° C. A solution of m-chloroperoxybenzoic acid (192mg) in methylene chloride (4 mL) was added dropwise. The mixture waswashed with aqueous sodium thiosulfate and with aqueous sodiumbicarbonate, dried (sodium sulfate) and the solvent removed underreduced pressure. The residue was chromatographed to give 660 mg ofintermediate Z.

Example 26

Compound 1

To a solution of tetrakis(triphenylphosphine)palladium (0) (166 mg) in 2mL of tetrahydrofuran: acetic acid (10:1) mixture was added a solutionof intermediate Z (660 mg) in 3 mL of the same solvent mixture. After 30minutes, additional tetrakis(triphenylphosphine)palladium (0) was added.After an additional 11/2 hours, toluene was added and the solventremoved under reduced pressure. The mixture was purified bychromatography on diethylaminoethylcellulose. The purified mixture wasdissolved in 0.1N aqueous sodium hydroxide, filtered through a 0.45 μsterile filter and purified by HPLC on a YMC-Pack ODS-AP column to give130 mg of compound 1.

Analytical data for some of the compounds and intermediates made by themethods described above is given below:

Compound 1: ¹ H NMR (CD₃ OD) δ: 5.3 (1H, m), 4.6 (1, m), 4.0 (m, m), 3.9(1H, d), 3.7 (1H, t), 3.6 (1H, t), 3.4 (3H, s), 3.3 (3H, t), 2.6 (2H,t), 2.3 (2H, m), 2.0 (2H, m), 1.7-1.2 (m, m), 0.9 (6H, t).

³¹ P NMR (CD₃ OD) δ:4.71, 3.98.

Compound 1: (M+Na)⁺ =1333

Compound 2: (M+3 Na)⁺ =1363

Compound 3: (M+3 Na)⁺ =1365

Compound 5: (M+Na)⁺ =1303

Compound 6: (M+Na)⁺ =1359

Compound 7: (M+Na)⁺ =1305

Compound 8: (M+3 Na)⁺ =1393

Compound 10: (M+Na)⁺ =1425

Intermediate G: ¹ H NMR (CDCl₃) δ: d, (1H), 3.9-3.7 (m, multiple), 3.65(t, 1H), 3.37 (s,3H), 3.2 (m,2H), 1.75 (q, 2H), 1.52 (s,3H), 1.4 (s,3H),1.3 (broad m,multiple), 0.95 (s,9H), 0.9 (t,3H), and 0.2 (d,6H)

Intermediate H: ¹ H NMR (CDCl₃) δ:4.58 (d,1H), 4.09 (m,2H), 3.9 (dd,1H),3.75 (dd,1H), 3.7 (m,1H), 3.5 (t,1H), 3.37 (s,3H), 3.23 (t,1H), 3.05(t,1H), 1.8 (m,2H), 1.68 (m,1H), 1.5 (m,1H), 1.3 (broad m,multiple),0.95 (s,9H), 0.9 (t,3H), 0.2 (d,6H).

Intermediate I:¹ H NMR (CDCl₃) δ:4,52 (d,1H), 4.05 (m,2H), 3.75 (m,1H),3.67 (t,1H), 3.5 (t,1H), 3.45 (s,3H), 3.35 (s,3H), 3.25 (t,1H), 3.05(t,1H), 1.8 (m,2H), 1.65 (m,1H), 1.5 (m,1H), 1.3 (broad s,m), 0.95(s,9H), 0.9 (t,3H), 0.2 (s,6H)

IntermediateJ:¹ H NMR (CDCl₃) δ:5.95 (m,2H), 5.35 (d,1H), 5.22 (d,1H),4.6 (q,2H), 4.5 (d,1H), 4.32 (q,1H), 3.9-3.75 (m,3H), 3.7 (dd,1H), 3.65(dd,1H), 3.45 (m,1H), 3.38 (s,3H), 3.33 (s,3H), 3.27 (t,1H), 3.2 (t,1H),1.9-1.75 (m,3H), 1.5 (m,1H), 1.3 (broad m,multiple), 0.95 (s,9H), 0.9(t,3H), 0.2 (s,6H)

Intermediate L:¹ H NMR (CDCl₃) δ:5.95 (d,1H), 5.4 (d,2H), 5.25 Z(d,2H),4.95 (d,1H), 4.7 (q,2H), 4.55 (q,2H), 4.32 (q,1H), 3.9-3.75 (m,3H), 3.7(dd,1H), 3.65 (dd,1H), 3.55 (m,1 H), 3.4 (m,1H), 3.4 (s,3H), 3.3 (s,3H),3.25 (m,1H), 1.75 (m,multiple), 1.5-1.4 (m,2H), 1.3 (broad s,multiple),0.95-0.9 (broad s,12H), 0.2 (d,6H)

Intermediate M: ¹ H NMR (CDCl₃) δ:5.95 (m,2H), 5.75 (d,1 H), 5.4 (d,1H), 5.25 (d,2H), 4.75-4.65 (dd,2H), 4.6 (q,1 H), 4.3 (q,1 H), 4.1(m,2H), 3.9 (m,2H), 3.65 (m,1 H), 3.4 (s,3H), 3.25 (s,3H), 1.75 (broadm,2H), 1.55-1.4 (m,2H), 1.3 (broad s,multiple), 0.9 (t,3H)

Intermediate O; ¹ H NMR (CDCl₃) δ: 4.5 (d,1 H), 3.8 (dd,1 H), 3.78(m,2H), 3.6 (m,multiple), 3.2 (m,2H), 1.5 (s,3H), 1.4 (s,3H), 1.3 (broads, multiple), 0.95 (s,9H), 0.9 (t,3H), 0.18 (d,6H)

Intermediate P:¹ H NMR (CDCl₃) δ:4.5 (d,1H), 3.75 (dd,2H), 3.6 (q,2H),3.5 (t,1H), 3.3 (m,1H), 3.2 (t,1H), 3.0 (t,1H), 1.6 (m,2H), 1.25 (broads,multiple), 0.95 (s,9H), 0.9 (t,3H), 0.18 (d,6H)

Intermediate Q: ¹ H NMR (CDCl₃) δ:4.5 (d,1H), 3.82 (t,2H), 3.7 (m,2H),3.6 (t,1H), 3.3 (m,1H), 3.2 (t,1H), 3.05 (q,2H), 1.6 (m,2H), 1.3 (broads,multiple), 0.95 (s,9H), 0.88 (s,9H), 0.85 (t,3H), 0.2 (d,6H), 0.1(d,6H)

Intermediate R: ¹ H NMR (CDCl₃) δ: 5.9 (m,1H), 5,4-5.25 (dd,2H), 4.75(t,1H), 4.6 (d,2H), 4.45 (d,1H), 3.75 (q,1H), 3.7 (d,2H), 3.53 (q,1H),3.38 (m,1H), 3.25 (t,1H), 3.15 (t,1H), 1.5 (t,2H), 1.25 (s, multiple),0.95(s,9H), 0.85 (m,12H), 0.2 (s,6H), 0.07 (s,6H)

Intermediate S:¹ H NMR (CDCl₃) δ: 5.9 (m,1H), 5.4-5.25 (dd,2H), 4.75(t,1H), 4.6 (d,2H), 4.52 (d,1H), 3.7 (m,multiple), 3.65-3.6 (dd,2H),3.55 (q,1H), 3.4 (m,1H), 3.28 (t,1H), 3.2 (t,1H), 1.5 (t,2H), 1.3 (s,multiple), 0.9 (s,9H), 0.85 (t,3H), 0.2 (s,6H)

Intermediate T: ¹ H NMR (CDCl₃) δ: 5.9 (m,3H), 5.6 (d,1H), 5.4-5.2(m,6H), 4.8 (d,1H), 4.7-4.6 (m,2H), 4.55 (q,1H), 4.5 (d,1H), 4.3 q,1H),3.8-3.7 (m,multiple), 3.6 (dd, 1H), 3.5 (m,multiple), 3.35 (s,3H), 3.2(s,3H), 3.15 (t,1H), 1.7 (m,2H), 1.5 (m,2H), 1.3 (s,multiple), 0.95(t,6H), 0.2 (t,6H)

Intermediate V: ¹ H NMR (CDCl₃) δ: 7.3 (d,1H), 5.95 (m,3H), 5.6 (d,1H),5.4-5.2 (m,6H), 4.95 (d,1H), 4.8 (d,1H), 4.7-4.5 (m,multiple)4.3 (q,1H),3.9-3.65 (m,multiple), 3.6 (m,multiple), 3.45 (t,1H), 3.4 (t,3H), 3.35(s,2H), 3.28 (3H), 2.5 (t,2H), 1.8 (m,2H), 1.6 (m,2H), 1.45 (m,2H), 1.3(broad s,multiple), 0.95-0.8 (m,18H), 0.15 (d,6H) Intermediate X: ¹ HNMR (CDCl₃) δ: 7.3 (d,1H), 5.95 (m,4H), 5.4-5.2 (m,8H), 4.95 (d,1H), 4.8(d,1H), 4.7 (t,1H), 4.6 (d,1H), 4.55 (q,1H), 4.3 (q,1H), 4.1 (t,1H), 3.9(q,1H), 3.8 (t,1H), 3.7-3.5 (m,multiple), 3.45 (t,1H), 3.35 (s,3H), 3.3(s,2H), 3.28 (s,3H), 2.5 (t,2H), 2.2 (t,1H), 2 (d,1H), 1.7 (q,2H), 1.6(m,2H), 1.3 (s,multiple), 0.950.8 (m,21), 0.15 (d,6H)

Intermediate Y: ¹ H NMR (CDCl₃) δ: 6.65 (d,1H), 6.55 (d,1H), 5.905(m,5H), 5.7 (m,1H), 5.4-5.2 (m,12H). 4.8 (m,2H), 4.6 (d,1H), 4.5(m,10H), 4.3 (q,1H), 4.1 (m,1H), 3.85-3.45 (m,multiple), 3.4 (s,3H),3.35 (s,3H), 3.25 (s,3H), 3.2 (t,1H), 2.5 (dd,2H), 2.2 (t,2H), 2(m,mutiple), 1.7-1.2 (m,mutiple), 0.9 (t,12H).

BIOLOGICAL EXAMPLES

Both bacterial LPS and bacterial lipid A elicit production of tumornecrosis factor (TNF), IL-1β, IL-6 and IL-8 as well as other cytokinesand cellular mediators in human whole blood and in a human macrophagecell lines. Generation of pathophysiological quantities of thesecytokines has been found to play an important part in the initiation ofthe systemic inflammatory response syndrome and septic shock. Theliposaccharide analogs described herein inhibit such LPS-and/or lipidA-mediated induction of cytokines as demonstrated by the followingexperiments.

Example A

In Vitro Inhibition of LPS-Induced Production of Cytokines

Whole human blood was prepared and tested as described (Rose et al.(1995) Infection and Immunity, 63 833-839). HL-60 cells were cultured inRPMI medium supplemented with 10% fetal calf serum and antibiotics, andinduced to differentiate into macrophages by treatment with 0.1 μM1,25-dihydroxycholecalciferol (Vitamin D3; Biomol Research Laboratories,Plymouth Meeting, Pa.), and tested for LPS mediated generation of IL-8.Briefly, bacterial LPS (i.e., from E. coli 0111:B4; Sigma Chemicals, St.Louis, Mo.) at 10 ng/mL or lipid A (Daiichi Chemicals, Tokyo, Japan)were added as 10-fold concentrated solutions in Ca⁺⁺, Mg⁺⁺ free Hank'sbalanced salt solution (CMF-HBSS; Gibco). In experiments involvinganalogs of the present invention, the analog was added immediatelybefore addition of LPS or lipid A in CMF-HBSS (e.g., between 0 and 100μM as a 10× concentrated aliquot). Following incubation of three hours,plasma was prepared from whole blood, or culture supernatant was removedand assayed for the presence of the indicated cytokine using an ELISAassay kit from Genzyme (Cambridge, Mass.) following the instructions ofthe manufacturer, however, any other standard ELISA kits may beutilized. Experiments were performed in triplicate at least twice.

The lipid A analogs inhibited LPS-induced production of TNF in humanwhole blood in a concentration-dependent manner. Of the analogs tested,Compound 1 was found to be one of the most effective compounds. Theresults of this test are as shown in FIG. 1. Compound 1 inhibitsLPS-induced production of TNF, exhibiting an IC₅₀ of approximately 1.5nM. Other analogs found to inhibit LPS-induced TNF production includedcompound 2, compound 3, compound 4, compound 5, compound 6, compound 7,compound 8, compound 9, and compound 10. These compounds exhibited IC₅₀s of between 1.5 nM and 159 nM.

Compound 1 also inhibited LPS-mediated induction of IL-8 in HL-60 (humanmacrophage-like) cells. Inhibition of IL-8 generation was complete atconcentrations of 1 nM and greater Compound 1 when either LPS or lipid Awas used as agonist.

Compounds of this invention similarly inhibited the LPS-inducedproduction of other cytokines in human whole blood, even though some ofthese cytokines were generated several hours after addition of LPS. Forinstance, IL-1β, and IL-6 requires four or more hours for maximum levelsto be reached, while IL-8 reaches maximum levels ten or more hours afterLPS addition. Using methods described above, compounds of this inventionwere added at a concentrations between 0 and 10 μM and LPS was added at10 ng/mL. Inhibition of production of TNF, IL-1β, IL-6, and IL-8 wasmeasured as a function of time after addition of LPS. This inhibition ofcytokine generation was also found to be concentration dependent, but inall cases, suppression of all cytokine synthesis was >90% at compound 1concentrations of 10 nM and more for up to 24 hours after addition ofLPS.

Example B

Persistence of compounds in human whole blood

Although some of the compounds of this invention are not rapidly removedfrom the circulation, their activity diminishes with a half life of 1-3hours. In order to maintain antagonistic efficacy, this rapiddeactivation may require continuous administration. The study of thisdeactivation has led to the development of an assay to measure in vitrodeactivation of drugs in human whole blood. This is done bypreincubating lipid A antagonists with blood for various periods of timefollowed by addition of the LPS "challenge" as described above,incubation for three hours, and assay for released cytokines. Aschematic for this assay is shown in FIG. 2.

Using this assay, it can be demonstrated that B531, as described byChrist, et al., U.S. application Ser. No. 07/935,050, "deactivates"(loses activity with increasing time of preincubation). As shown in FIG.3, compound 1 also deactivates, but its superior activity and decreaseddeactivation rate makes it as active after 6 hours as B531 was justafter its addition. These data are the average of 7 separate experimentsrun in triplicate.

Example C

Inhibition of TNF or IL-6 Production in in vitro animal model systems

LPS-induced TNF or IL-6 production was inhibited by compounds of thepresent invention in whole blood or macrophages isolated from guineapigs, rats and mice. Hartley-White guinea pig (Elm Hill Breeders,Chelmsford, Mass.) and C57BU6 mouse (Jackson Labs, Bar Harbor, Me.)macrophages were isolated from the abdomen of primed animals. Primingwas accomplished by intraperitoneal injection of 2 mg of Bacilluscalmette guerin(BCG; RIBI Immunochemical Research, Inc., Hamilton,Mont.) at a concentration of 10 mg/mL in physiological saline for miceand 2 mg of BCG at a concentration of 2 mg/7 mL in mineral oil forguinea pigs. Three days post-injection, peritoneal macrophages wereisolated from the abdomen of the animals by standard techniques. Cellswere allowed to adhere to culture plates for two to three hours and werethen cultured with RPMI 1640 medium containing 10% fetal calf serum andLPS (final concentration of 10 ng/mL) was added as described above. Totest inhibition, compounds of this invention (at a concentration ofbetween 0 and 100 μM) were added to the culture medium just prior to LPSaddition. Following a three-hour incubation period, guinea pig, mouseand rat TNF levels and/or IL-6 levels were assayed by ELISA, or by thecytolytic bioassay described in Lymphokines 2:235, 1981 for TNF releasedfrom guinea pig macrophages. In mouse peritoneal macrophages, Compound 1provided effective inhibition (IC₅₀ =16 nM for IL-6 and 20 nM for TNF,respectively); in guinea pig macrophages, the IC₅₀ for TNF release was0.3 nM and in rat peritoneal macrophages, the IC₅₀ for release of TNFwas 11 nM.

Example D

In Vivo Assays

BCG-primed mice (Vogel, S. et al. J. Immunology 1980, 124, 2004-2009)were utilized as an in vivo assay system for monitoring the inhibitoryeffects of lipid A analogs on (1) LPS-induced TNF production and (2)LPS-induced lethality as follows.

Five week old male C57BL/6 mice (supra) were primed by intravenous tailvein injection with 2 mg of BCG. Ten days post-injection, E. coli LPS(supra) in pyrogen-free 5% glucose solution (Otsuka PharmaceuticalsInc., Tokyo, Japan) was administered intravenously through the tail veinof the BCG-primed mice. LPS was administered at 1-3 μg/mouse for bothTNF production and mortality studies. The test compound was administeredas a component of the injected LPS solution at a concentration ofbetween 3 and 300 μg/mouse. Plasma was obtained one hour post LPSinjection, and TNF was assayed by the ELISA assay described above.Mortality resulting from septic shock was recorded for 36 hours afterLPS injection.

Compounds of this invention effectively suppressed the production of TNFfollowing administration of LPS. Compound 10 and Compound 1 effectivelyinhibited TNF production in vivo in mice (ED_(50s) =5 and 10.6 μg/mouse,respectively). Compound 2, compound 3, compound 4, compound 5, compound6, compound 7, compound 8, and compound 9 also inhibited TNF productionwith ED₅₀ s between 10 and 200 μg/mouse with compounds 5, 6, and 7giving ED₅₀ values >100.

In parallel experiments carried out in guinea pigs, these analogs werealso effective inhibitors of LPS-induced TNF production in vivo (optimumED₅₀ 's between 2.3 and 6.1 μg/guinea pig for compound 1, compound 7,and compound 10.

Therapy

The lipid A analogs described herein provide useful therapeutics for thetreatment or prevention of any LPS-mediated inflammation or disorder.Such disorders include without limitation: endotoxemia (or sepsissyndrome) resulting from a gram negative bacteremia (with itsaccompanying symptoms of fever, generalized inflammation, disseminatedintravascular coagulation, hypotension, acute renal failure, acuterespiratory distress syndrome, hepatocellular destruction, and/orcardiac failure); and LPS-mediated exacerbation of latent or activeviral infections (e.g., infection with HIV-1, cytomegaloviruses, herpessimplex, and influenza virus).

The lipid A analog is typically administered in a pharmaceuticallyacceptable formulation, e.g., dissolved in physiological saline (whichmay include 5% glucose). When the lipid A analog is provided for thetreatment of a viral infection, it may be administered in conjunctionwith appropriate viricidal agents. The Lipid A analog may be stored as afreeze-dried formulation.

Lipid A analogs are administered in dosages which provide suitableinhibition of LPS activation of target cells; generally, these dosagesare, preferably, between 0.01-50 mg/patient/day, more preferably,between 0.05-25 mg/patient/day, and most preferably, between 1 and 12mg/patient/day.

The drug should be injected or infused as soon as possible when SIRS canbe diagnosed using clinical predictors such as the APACHE score (Knaus,et al., 1991 Chest 100: 1619-36 and Knaus, et al., 1993 JAMA: 1233-41)or other clinical predictors. In addition, injection or infusion shouldcommence as soon as possible after exposure to endotoxin or diagnosis ofsystemic gram negative bacterial infection, especially if a more rapidor early diagnostic indicator of systematic gram negative infectionbecomes available.

Prophylactic indications for use of these drugs may include their usewhen exposure to endotoxin can be anticipated. This can occur when:

1) there is an increased probability of elevation of systemic(blood-borne) endotoxin from systemic or localized gram negativebacterial infection (such as during surgery);

2) there is an increased probability that blood levels of endotoxin mayincrease. In the normal physiological state, endotoxin only minimallytranslocates across the gut endothelium into splanchnic circulation.This translocated endotoxin is usually then cleared by the liver (andpossibly other cells and organs). Increases in blood endotoxin levelscan occur when the rate of endotoxin clearance by the liver (or otherendotoxin sequestering cells or organs) decreases. Augmentation of guttranslocation can occur after gut ischemia, hypoxia, trauma, or otherinjury to the integrity of the gut lining (or by drug or alcoholtoxicity). Blood levels of endotoxin increase when liver function iscompromised by disease (cirrhosis), damage (surgery or trauma), ortemporary removal (as during liver transplantation);

3) there is an acute or chronic exposure to externally-derived endotoxinresulting in inflammatory response; this exposure can be caused byinhalation or other means of uptake of endotoxin. One example ofSIRS-inducing endotoxin uptake is corn dust fever (Schwartz, et al.,1994 Am J Physiol. 267: L609-17), which affects workers in the grainindustry, for example, in the American mid-west. Such workers can beprophylactically treated, e.g., daily, by inhaling an aerosolizedformulation of the drug prior to beginning work in, e.g., fields orgrain elevators.

For most other prophylactic and therapeutic applications, IV infusion orbolus injection will be used. Injection is most preferable, but infusionmay be warranted in some cases by pharmacokinetic requirements.

The treatment should be initiated as soon as possible after diagnosis ofSIRS, and should continue for at least three days, or when assessment ofrisk of mortality is reduced an acceptable level.

What is claimed is:
 1. A compound of the formula: ##STR75## where R¹ isselected from the group consisting of ##STR76## where each J, K and Q,independently, is straight or branched C1 to C15 alkyl; L is O, NH, orCH₂ ; M is O or NH; and G is NH, O, S, SO, or SO₂ ;R² is straight orbranched C5 to C15 alkyl; R³ is selected from the group consistingofstraight or branched C5 to C18 alyl, ##STR77## where E is NH, O, S,SO, or SO₂ ; each A, B and D, independently, is straight or branched C1to C15 alkyl; R⁴ is selected from the group consisting ofstraight orbranched C4 to C20 alkyl, and ##STR78## where each U and V,independently, is straight or branched C2 to C15 alkyl and W is hydrogenor straight or branched C1 to C5 alkyl; R_(A) is R⁵ or R⁵ --O--CH₂ --,R⁵ being selected from the group consisting of hydrogen, J', J'--OH,--J'--O--K',--J'--O--K'--OH, and --J'--O--PO(OH)₂, where each J' and K',independently, is straight or branched C1 to C5 alkyl; R⁶ is selectedfrom the group consisting of hydroxy, halogen, C1 to C5 alkoxy and C1 toC5 acyloxy; A¹ and A², independently, are selected from the groupconsisting of ##STR79## where Z is straight or branched C1 to C10 alkyl;or a pharmaceutically acceptable salt thereof.
 2. compound of claim 1with the formula: ##STR80## where R¹ is selected from the groupconsisting of ##STR81## where each J, K and Q, independently, isstraight or branched C1 to C15 alkyl; L is O, NH, or CH₂ ; M is O or NH;and G is NH, O, S, SO, or SO₂ ;R² is straight or branched C5 to C15alkyl; R³ is selected from the group consisting ofstraight or branchedC5 to C18 alyl, ##STR82## where E is NH, O, S, SO, or SO₂ ; each A, Band D, independently, is straight or branched C1 to C15 alkyl; R⁴ isselected from the group consisting ofstraight or branched C4 to C20alkyl, and ##STR83## where each U and V, independently, is straight orbranched C2 to C15 alkyl and W is hydrogen or straight or branched C1 toC5 alkyl; R⁵ is selected from the group consisting of hydrogen,J',--J'--OH, --J'--O--K',--J'--O--K'--OH and --J'--O--PO(OH)₂, whereeach J' and K', independently, is straight or branched C1 to C5 alkyl;R⁶ is selected from the group consisting of hydroxy, halogen, C1 to C5alkoxy and C1 to C5 acyloxy; A¹ and A², independently, are selected fromthe group consisting of ##STR84## where Z is straight or C1 to C10alkyl; or a pharmaceutically acceptable salt thereof.
 3. A compound ofclaim 2 where R² is C8 to C15 straight or branched alkyl.
 4. A compoundof claim 2 where R² is C9 to C12 straight or branched alkyl.
 5. Acompound of claim 2 where R² is C10 straight or branched alkyl.
 6. Acompound of claim 2 where A¹ and A², independently, are OH or--O--PO(OH)₂.
 7. A compound of claim 2 where R⁶ is hydroxy.
 8. Acompound of claim 2 where R⁵ is C1 to C5 straight or branched alkyl. 9.A compound of claim 2 where R¹ is selected from the group consisting of##STR85## where each J, K and Q, independently, is straight or branchedC1 to C15 alkyl.
 10. A compound of claim 2 where R⁴ is selected from thegroup consisting ofstraight or branched C4 to C20 alkyl, and ##STR86##where U is straight or branched C2 to C5 alkyl, V is straight orbranched C5 to C12 alkyl, and W is hydrogen or straight or branched C1to C5 alkyl.
 11. A compound of claim 2 having the following structure##STR87## or a pharmaceutically acceptable salt thereof.
 12. A compoundof claim 2 having the following structure ##STR88## or apharmaceutically acceptable salt thereof.
 13. A method for treatingsepsis in a patient, said method comprising administering compound ofclaim 1 to said patient by bolus injection.